SUMMARY Hepadnaviruses (hepatitis B viruses) cause transient and chronic infections of the liver. Transient infections run a course of several months, and chronic infections are often lifelong. Chronic infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. The replication strategy of these viruses has been described in great detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. Studies on how the virus evades the immune response to cause prolonged transient infections with high-titer viremia and lifelong infections with an ongoing inflammation of the liver are still at an early stage, and the role of the virus in liver cancer is still elusive. The state of knowledge in this very active field is therefore reviewed with an emphasis on past accomplishments as well as goals for the future.

Human hepatitis B virus (HBV) is the prototype of a family of small DNA viruses that productively infect hepatocytes, the major cell of the liver, and replicate by reverse transcription of a terminally redundant viral RNA, the pregenome. Upon infection, the circular, partially double-stranded virion DNA is converted in the nucleus to a covalently closed circular DNA (cccDNA) that assembles into a minichromosome, the template for viral mRNA synthesis. Infection of hepatocytes is non-cytopathic. Infection of the liver may be either transient (<6 months) or chronic and life long, depending on the ability of the host immune response to clear the infection. Chronic infections can cause immune mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of carcinogenesis are unclear. Antiviral therapies with nucleoside analog inhibitors of viral DNA synthesis delay sequelae, but cannot cure HBV infections due to the persistence of cccDNA in hepatocytes.

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-␣) and ribavirin combination therapy. The mechanism of the IFN-␣ response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-␣ reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-␣ independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-␣. Finally, under our conditions, selection for IFN-␣-resistant variants did not occur.Hepatitis C virus (HCV) causes persistent infection in approximately 80% of infected adults and variable and severe liver disease in an estimated 70% of those who cannot clear the virus (1). HCV is an enveloped, positive-stranded RNA virus encoding a polyprotein that is proteolytically processed into 10 polypeptides. Four of them are enzymes: cysteine and serine proteases, an ATP-dependent helicase, and an RNA-directed RNA polymerase (17). While these enzymes are used as potential targets for virus-specific antiviral therapies, their genes exhibit high variability among the different HCV genotypes, and, most likely, drug-resistant variants will evolve during antiviral therapy. Currently available combination therapy with alpha interferon (IFN-␣) and ribavirin is effective in less than 50% of treated patients. Although the mechanism controlling the IFN response in patients is likely to be complex, there is evidence that nonstructural (NS) protein 5A (NS5A) evolves to confer resistance against IFN-␣ during antiviral therapy (5, 6). This resistance is believed to be a consequence of a specific interaction between NS5A and protein kinase R (PKR), an important mediator of the antiviral program induced by IFN-␣.Unfortunately, efforts to investigate the molecular mechanisms responsible for IFN resistance were hampered by the lack of tissue culture systems permissive for the replication and production of infectious HCV from available cDNA clones. Recently, Lohmann and colleagues (13) reported that a subgenomic replicon containing a neomycin phosphotransferase gene (neoR) in lieu of the viral structural genes replicated in Huh7 cells (see Fig. 1A). However, from the low frequency with which Huh7 cells supported replication, it is possible that the selected isolate is defective and requires genetic changes for efficient genome synthesis. This possibili...

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